1. Field of the Invention
This invention relates to DNA comprising genetic information for L-.alpha.-glycerophosphate oxidase, a transformant possessing said DNA, a polypeptide produced by causing said transformant to express the genetic information of said DNA, and a process for producing said L-.alpha.glycerophosphate oxidase.
2. Description of the Background
L-.alpha.-glycerophosphate oxidase is an enzyme catalyzing an enzymatic reaction for producing dihydroxyacetone phosphate and one mole of hydrogen peroxide from L-.alpha.glycerophosphate and one mole of oxygen according to the following reaction formula: EQU L-.alpha.-glycerophosphate+O.sub.2 .fwdarw.dihydroxyacetone phosphate+H.sub.2 O.sub.2
Since L-.alpha.-glycerophosphate oxidase (hereinafter abbreviated as "GPO") is an oxidase having as its substrate L-.alpha.-glycerophosphate, as can be seen from the above reaction formula, it can be used for the quantitative determination of L-.alpha.-glycerophosphate. In addition, this enzyme, when used in combination with other enzymes such as lipase, glycerol kinase, or the like, or with an ATP reagent, provides a simple and specific determination method for the components involving the reaction, such as lipase activity, triglyceride, glycerine, ATP, and the like. Because of these reasons, L-.alpha.-glycerophosphate oxidase is a major enzyme for biochemical quantitative analysis which is to replace conventional, non-specific chemical quantitative analysis. Thus, the enzyme is very useful in the field of clinical diagnosis as well as in the field of research.
GPO has long been known to exist in the natural world. For instance, the enzyme has been reported to exist in microorganisms belonging to genera such as Streptococcus, Lactobacillus, Leuconostock, Pediococcus, and Aerococcus [Archives of Biochemistry and Biophysics, 88, 250 (1960); Japanese Patent Laid-open No. 72,892/1978; and Japanese Patent Laid-open No. 15,746/1980].
However, the production of GPO by these conventionally known GPO producing-microorganisms has had several problems. Specifically, all of the known GPO producing-microorganisms achieved only a low GPO productivity. GPO producing-microorganisms belonging to the strain Streptococcaceae and the strain Lactobacilleceae require the addition of a substance inducing the production of GPO, such as glycerol, ascorbic acid, .alpha.-keto acid, or the like. In addition, the elimination of other enzymes and the like which exist together with GPO in the product of these microorganisms is very difficult. Because of these problems a high quality GPO could only be produced at an extremely high cost, and this has thus prevented GPO from being widely used as a reagent for research purposes and for clinical diagnosis.
In addition, there have been no reports concerning the detailed primary structure of the GPO gene, nor about the primary structure of the amino acid sequence for the protein constituting GPO.
The present inventors have undertaken extensive studies for promoting the productivity of GPO and for reducing the amount of other co-existing enzymes (contaminant enzymes) contained in the product. As a result, the inventors have succeeded in separating the GPO gene and in the determination of its primary structure. The inventors have further established, through the application of genetic engineering techniques, a process for producing GPO at a high productivity and without using any additives to induce GPO production in the production medium.